The goal of this project is to study the molecular pathogenesis of recessive osteogenesis imperfecta (OI)- a brittle bone disease with craniofacial abnormalities. Morello et. al. first identified mutations in Cartilage Associated Protein (CRTAP) as causative of OI Type VII, a recessive OI[3]. Additionally, mutations to prolyl 3- hydroxylase (P3H1) have been identified as causative of recessive OI[4]. CRTAP and P3H1 form a complex with cyclophilin B (CypB) and this complex functions to 3-hydroxylate a single proline residue on the alpha 1 chain of type I collagen[2]. In addition to its function as a 3-hydroxylase, CypB functions as a cis/trans isomerase (PPIase)[9]. Many of the amino acid residues present in the triple helix region are in the cis form and collagen will not fold properly unless all bonds are in the trans conformation[11]. Thus, the function of PPIase is essential for proper collagen folding. Interestingly, the inhibition of CypB by cyclosporin A results in overmodified collagen and a delay in collagen secretion- similar to the collagen defects observed in recessive OI patints[19]. Recently, data suggest that the presence of 3-hydroxyproline may stabilize the helix domain[7]. However, its presence or absence is not likely to affect the structural integrity of collagen, but its absence may disrupt protein-protein interactions integral for proper collagen folding[7]. Preliminary data demonstrate that P3H1 and CRTAP stabilize each other. Thus, when one complex member is mutant, the other is degraded. Since all identified CRTAP and P3H1 mutations are hypomorphic or null, it is unclear whether the 3- hydroxylation function and/or the PPIase function are important in the molecular pathogenesis of recessive OI. Thus, this project aims to study the contribution on P3H1 hydroxylase activity and how it pertains to the pathogenesis of recessive OI by inactivating the hydroxylase domain while maintaining the protein structure. To address the contribution of P3H1 hydroxylase activity, the following experiments are proposed: 1)study the consequences of 3-hydroxylase inactivation on collagen secretion, and modification by cell culture, and 2) assess whether 3-hydroxylation contributes to an osteoporotic phenotype by studying knock-in mice containing the point mutation demonstrated to best inactivate the hydroxylase function while maintaining the stability of the P3H1 complex. Public Health Relevance: This project dissects the role of the enzymatic activity of the P3H1 protein within a collagen modifying complex that serves two roles in the processing of collagen. The results of this project will impact our understanding of the regulation of collagen processing and will shed light on the molecular abnormalities leading to recessive OI.